This invention relates to the field of bacterial strains used for bioremediation. In particular, it relates to endospore-forming bacterial strains that can degrade polyaromatic hydrocarbons, and methods to isolated these bacterial strains and to use these strains for bioremediation.
Various scientific and scholarly articles are referred to throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
Polyaromatic hydrocarbons (PAHs) are widespread pollutants, particularly in conjunction with the marine environment. High PAH levels are known to be toxic, mutagenic and carcinogenic, and therefore pose a considerable threat to the public health. Many microorganisms degrade PAHs, and there is a strong interest in applying bioremediation approaches to remove PAHs from the environment.
Bacteria that degrade 2-3 ring low molecular weight polyaromatic hydrocarbons, such as naphthalene, phenanthrene, bi-phenyl and fluorene, are taxonimically diverse. Gram negative genera such as Pseudomonas, Burkholderia, Alcaligens, Sphingomonas, Vibrio and Comamonas are common. While less common, the gram-positive species such as Mycobacterium, Nocardia, Rhodococcus and Gordona are also known to degrade low molecular weight PAHs.
Microorganisms that can degrade the 4-ring and higher high molecular weight PAHs such as pyrene, fluoranthene and benz[a]anthracene are much rarer. The degradation of high molecular weight PAHs is much slower and less extensive than the degradation of low molecular weight PAHs in the environment.
The isolation of new genera of PAH-degrading bacteria is sought, particularly species with novel combinations of growth and degradative characteristics. In particular, species are sought with a broad range of PAH specificities. Species that can degrade the higher molecular weight PAHs are particularly valuable. Finally, PAH-degrading species that are capable to surviving adverse nutritional and environmental conditions by forming endospores are of particular interest.
The invention pertains to isolated strains of bacteria that degrade polyaromatic hydrocarbons, a method of isolating the strains of the invention and a method of using the strains of the invention for bioremediation. The bacterial strains are in the family Bacillaceae and are novel for their combination of PAH-degrading and endospore-forming properties. These bacterial strains have utility for bioremediation of polyaromatic hydrocarbon in contaminated environments.
The first aspect of the invention is an isolated strain in the family Bacillaceae that has broad PAH-degrading capabilities in that it degrades at least two of the PAHs naphthalene, phenanthrene and biphenyl. In a preferred embodiment, the strain additionally degrades pyrene when induced by phenanthrene. Preferably, the strain is in the isolate Paenibacillus, and most preferably in the species P. validus. This isolated bacterial strain may be isolates PR-P1, PR-N4 and PR-B2, which are ATCC Accession Nos. PTA-643 PTA-642 and PTA-641 respectively.
The second aspect of the invention is an isolated bacterial strain that is the newly described species xe2x80x9cPaenibacillus naphthalenovoransxe2x80x9d and degrades naphthalene. This bacterial strain is further described by several features. The strain has, in a preferred embodiment, a whole cell fatty acid composition in which 16:1 xcfx8911c fatty acids comprise at least 8.8%, and additionally, in a most preferred embodiment, at least 5.8% but not more than 10.0% 17:0 anteiso fatty acids, when grown on trypticase soy agar at 28xc2x0 C. for 24 hours prior to analysis. In another preferred embodiment, the isolated strain has a whole cell fatty acid similarity index as compared to isolate PR-N1 of greater than 0.4 when grown on trypticase soy agar at 28xc2x0 C. for 24 hours prior to analysis. In another preferred embodiment, the bacterial strain has a 16S rRNA gene sequence that is at least about 95% homologous to SEQ ID NO:8. In another preferred embodiment, the genomic DNA of the strain exhibits at least 10% binding to the genomic DNA of isolate PR-N1. In another preferred embodiment, the strain is ATCC Accession No. PTA-640.
Another aspect of the invention is a plurality of bacterial strains that degrade PAHs. This plurality comprises at least two of the following: Paenibacillus validus, a sphingomonad, an Arthrobacter, xe2x80x9cPaenibacillus naphthalenovoransxe2x80x9d and a nocardioform. In a preferred embodiment, the P. validus strain is ATCC Accession No. PTA-641, PTA-642 or PTA-640 and xe2x80x9cP. naphthalenovoransxe2x80x9d strain is ATCC Accession No. PTA-640.
Another aspect of the invention is a composition for bioremediation that comprises one of the aforementioned bacterial strains and an ecologically acceptable carrier.
Another aspect of the invention is a method for removing PAHs from a substrate comprising contacting the substrate with the bacterial strain of the invention for a time sufficient to remove the PAHs. In preferred embodiments, the substrate of the method is solid or liquid, with optional aeration. In a more preferred embodiment, the method additionally comprises planting plants in the substrate, and in a most preferred embodiment, the plant is Spartina alterniflora. In another most preferred embodiment, the method has the additional step of inducing of the bacterial strain with phenanthrene.
Another aspect of the invention is a method to isolate endospore-forming PAH-degrading bacterial strains. This method comprises the steps of growing a heterogenous culture with a PAH as the sole carbon source, and heating the culture to a temperature for a time sufficient to kill vegetative bacteria. In preferred embodiments, the PAHs used are naphthalene, phenanthrene or biphenyl. In another preferred embodiment, the culture is heated to 70 to 90xc2x0 C. for 10 to 20 minutes.
Other features and advantages of the present invention will be better understood by reference to the drawings, detailed description and examples that follow.